1. Field of the Invention
The present invention relates to plant genetic engineering, particularly to a cDNA encoding a Maize 33 kD cysteine proteinase and its use to transform cells to express the cysteine proteinase to provide insect resistance.
2. Discussion of the Background
The larvae of both fall armyworm and southwestern corn borer are serious insect pests of corn (Zea mays L.) in the southern United States and cause damage to plants by feeding on leaves within the whorls. Several germplasm lines with resistance to these two insects have been developed and released (Williams et al, Crop Sci., 22:1269-1270 (1982); Williams et al, Crop Sci., 24:1217 (1984); Williams et al, Crop Sci., 30:757 (1990)). These lines also show resistance to several additional lepidopterous insects, including the sugarcane borer (Diatraea saccharalis [Fab]), the corn earworm (Helicoverpa zea [Boddie]), and the European corn borer (Ostrinia nubilalis [Huber]) (Davis et al, Miss. Agric. For. Exp. Stn. Tech. Bull., 157:1-6 (1988)).
Fall armyworm and southwestern corn borer larvae feed more extensively on whorls of susceptible plants than they do on resistant plants. Thus, larvae recovered from resistant plants are smaller than those recovered from susceptible plants (Williams et al, Crop Sci., 29:913-915 (1989)), with both antibiosis and nonpreference appearing to be operative as mechanisms of resistance (Wiseman et al, J. Econ. Entomol., 74:622-624 (1981) and Wiseman et al, Protection Ecology, 5:135-141 (1983)). Similar results have been obtained using callus initiated from mature embryos of resistant and susceptible genotypes (Williams et al, Crop Sci., 23:1210-1212 (1983)). Attempts to identify specific secondary metabolites that may be involved in resistance have been inconclusive (Hedin et al, J. Agric. Food Chem., 32:262-267 (1984)).
Several endogenous plant proteins may confer resistance to insect pests. These include the Ser proteinase inhibitors, lectins, and .alpha.-amylase inhibitors (Shade et al, Biotechnology, 12:793-796 (1994)). Proteins such as these may not be highly toxic, but they can control the rate of development of pest insect populations (Ibid.). These moderately toxic proteins exert a less stringent selection pressure than highly toxic insecticides and slow the development of resistance (Tabashnik, Annu. Rev. Entomol., 39:47-49 (1994)).
Although there is little specific information about the role of proteinases in insect resistance, it is generally believed that hydrolytic enzymes are involved in the plant defense response (Boller, in "Plant Proteolytic Enzymes, Vol. I, M. J. Dalling, Ed., CRC Press, Boca Raton, Fla., (1986)). Typically, hydrolytic enzymes are sequestered in the vacuole and are released when it is broken. The released hydrolases may be the first line of defense against pathogen or herbivore attack (Ibid.). Some proteinases such as the one found in Phaseolus vulgaris leaves (Van der Wilden et al, Plant Physiol., 73:576-578 (1983)) are present in the cell wall and thus provide another site of defense. Feeding by Spodoptera littoralis induces Leu aminopeptidase in tomato (Pautot et al, Proc. Natl. Acad. Sci. USA, 90:9906-9910 (1993)). This proteinase may be involved in systemic response to wounding, which leads to the expression of plant defense proteins (Pearce et al, Science, 253:895-897 (1991)).
Cysteine proteinases are endoproteinases found in bacteria, eukaryotic microorganisms, plants and animals (Barrett, in "Plant Proteolytic Enzymes, Vol. I, M. J. Dalling, Ed., CRC Press, Boca Raton, Fla., pp. 1-16 (1986)). One of the most studied cysteine proteinases is papain, which is found in the latex of Carica papaya L (Ibid.). There is a large family of enzymes with similarity to papain, including the cathepsins present in mammalian lysosomes (Ibid.). The thiol group on cys-25 of papain is required for activity, hence the name cysteine proteinase. This class of enzymes is inhibited by iodoacetate, E64, and small proteins called cystatins (Ibid.).
Many cysteine proteinases have been identified in plants. One group, synthesized during seed germination, is involved in storage protein degradation. Several isozymes from this group have been purified and cloned from barley (Kohler et al, Plant Cell, 2:769-783 (1990)), corn (deBarros et al, Plant Science, 99:189-197 (1994) and Mitsuhashi et al, Plant Physiol., 104:401-407 (1994)), rice (Watanabe et al, J. Biol. Chem., 266:16897-16902 (1991)), and other species.
Although the nucleus of every cell contains the entire complement of genomic DNA, a cell in a given tissue does not express every single gene in the genome. Rather, a given cell expresses a population of genes which code for proteins that are critical for maintaining cellular physiology. The challenge every cell faces is to express thousands of different genes at exactly the right times and in exactly the right amounts to prevent cellular physiology from becoming compromised.
Tissue-specific gene expression is often regulated at the level of gene transcription. Transcription is the process by which double-stranded DNA is read into singlestranded RNA. The processes of transcription initiation, and regulation of subsequent changes in rates of transcription reinitiation, are controlled by protein-DNA interactions.
Transcription is a highly regulated process in which nuclear gene regulatory proteins (often called transcription factors) bind, with high affinity, to short (five to fifteen base pair) lengths of DNA sequence known as control elements. Protein-DNA interactions occur in the promoter, 5' flanking, and 3' flanking regions of the gene. Through such protein-DNA interactions, gene expression is programmed to respond to changes in extracellular signals such as light, temperature, growth factor concentration, hormone concentration, and drug concentration and post-translational modification of protein (level of phosphorylation, myristylation, etc.). Such transcriptional mechanisms regulate tissue-specific protein expression.